The applicant has identified a protein, Red 25, that binds to a site in the promoter for HMG CoA reductase required for feedback regulation by cholesterol. Since HMG CoA reductase is the rate controlling enzyme of cholesterol biosynthesis the study of a protein involved in its regulation is essential for understanding the molecular mechanisms involved in maintaining cholesterol homeostasis. The long-term plan is to characterize the Red 25 regulatory protein in detail and determine its exact role in modulating expression of the HMG CoA reductase gene. This information will be used in the future to study the role of Red 25 in cholesterol homeostasis and determine if it is involved in diseases of cholesterol accumulation such as coronary artery disease (CAD). The experiments described in this proposal are to isolate cDNA clones that encode the Red 25 protein and to initiate a thorough study of its mode of action. The protein will be expressed from the cDNA in E. coli for the production of large amounts for both functional studies and to prepare antisera directed against Red 25. Hybridization probes prepared from the cDNA will be used along with the Red 25 antisera to study the pattern of Red 25 mRNA and protein expression in whole animals and in cell lines that can be manipulated experimentally to modulate HMG CoA reductase expression over a 100 fold range. In the future, the human gene for Red 25 will be cloned and the segregation of mutant alleles will be followed in families with a genetic tendency to develop coronary artery disease. The identification of functional domains by the experiments described in the present proposal will be compared to the location of human mutations of the Red 25 gene to understand how a mutation that results in a specific effect in the laboratory assays correlates to a perturbation of cholesterol balance in humans.